May 11, 2022
ViriCan Protocol: PBMC isolation from whole blood
This protocol is a draft, published without a DOI.
- 1Universidade Estadual Paulista (UNESP)
Protocol Citation: Deilson Elgui De Oliveira 2022. ViriCan Protocol: PBMC isolation from whole blood. protocols.io https://protocols.io/view/virican-protocol-pbmc-isolation-from-whole-blood-b83rrym6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 09, 2022
Last Modified: May 11, 2022
Protocol Integer ID: 62289
Keywords: blood, mononuclear cells, PBMC isolation
Abstract
Protocol for isolation of peripheral blood mononuclear cells for downstream applications requiring viable cells or cell pellets to extract DNA or RNA.
Guidelines
Notes:
- From Thermofisher (Answer Id: E4470): "Blood collected with EDTA typically has the highest DNA contamination, blood collected with heparin typically has less than that collected with EDTA, and blood collected with citrate shows the least DNA contamination of the three. (Formulation for citrate solution: 3.8% (w/v) which is 3.8 g/100 mL of water. Use 0.5 mL for every 4.5 mL of blood. Rock gently back and forth after adding citrate solution to mix.) Adding 12 µL of 5 N acetic acid per milliliter of TRIzol Reagent may help, although there may still be a problem with DNA contamination. Using plasma or serum works best. The fresher the blood sample the better the RNA. Degraded RNA has been observed in blood that has been processed in as little as two hours after drawing."
Reagent manuals:
- Ficoll-Paque Plus: https://drive.google.com/file/d/1hlvoGTZ60NTTsKaTwFiznoF1eh4vufwm/
Materials
Recommended blood collection tubes :
Lavender cap, with EDTA (Ethylenediaminetetraacetic Acid)
Green cap, with Heparin (Sodium/Lithium/Ammonium)
PBMC Separation Reagents:
Ficoll Paque PLUSGe HealthcareCatalog #17144003-500 ml
Histopaque 1077Sigma AldrichCatalog #1077-1
Protocol materials
Ficoll Paque PLUSGE HealthcareCatalog #17144003-500 ml
Materials
Histopaque 1077Merck MilliporeSigma (Sigma-Aldrich)Catalog #1077-1
Materials
Blood colection
Blood colection
Collect 4 mL of whole blood in tubes with anticoagulant*
Note
(*) Collection tubes with EDTA (lavender cap) or heparin (green cap)
PBMC isolation
PBMC isolation
Add 3 mL of Histopaque/Ficoll to 15mL conical centrifuge tube
Note
Bring the tubes to Room temperature before use
Carefully layer about 3 mL of whole blood on top of the Histopaque/Ficoll reagent.
Note
Let the blood layer the separation reagent by pipetting it on the wall of the conical tube with a Pasteur pipette or 1mL pipette tip.
Centrifuge Room temperature at 400 x g, 00:30:00 ,
Note
Centrifugation at lower temperatures (e.g., 4°C) may result in cell clumping and poor recovery.
Carefully transfer the middle opaque phase containing the PBMC to a new identified tube as follows and proceed to obtain PBMC's lisates or viable PBMCs for cellular assays.
Note
For DNA/RNA extraction: nuclease-free 2mL microcentrifuge tube. Go to procedure starting in step 6
To isolate viable PBMC: 15mL centrifuge conical tubes. Go to the procedure starting Step 8
(Optional) PBMC lysis to extract DNA/RNA
(Optional) PBMC lysis to extract DNA/RNA
Pellet the cells by centrifugation 1000 x g, 4°C, 00:05:00
Note
Washing cells before addition of TRIZOL® Reagent should be avoided as this increases the possibility of mRNA degradation
5m
Add To the PBMCs pellet:
250 µL of TRIZol LS Reagent
(General recommendation: 1 mL of TRIZol per 5-10 ×10e6 eukaryotic cells)
Homogenize to lysate the cells
Note
NOTE: According to ThermoFisher, "After homogenization and before the addition of chloroform, samples can be stored at -60 to -70°C for at least one month."
Store PBMC lisates in Trizol at -70 °C (ULT Freezer) or immediately proceed to nucleic acid isolation
Note
RNA extraction with Trizol: https://www.protocols.io/view/total-rna-extraction-from-pbmc-with-trizol-reagent-b83trynn
(Optional) PBMC clearing
(Optional) PBMC clearing
10m
10m
To the 15mL conical centrifuge tubes, add 10 mL of On ice isotonic 1X PBS (Phosphate-buffered saline )
Centrifuge 250 x g, 00:10:00
10m
Aspirate the supernatant and discard
Wash the PBMC pellet
Resuspend the cell pellet with 5.0 mL isotonic 1X PBS (Phosphate-buffered saline )
Mix by gentle aspiration with a Pasteur pipet or pipetting up and down with 1mL tip
Centrifuge 250 x g, 00:10:00
10m
Discard supernatant
Repeat the PBMC wash procedure twice
Proceed to downstream applications with the isolated PBMCs